In conjunction with our studies on mammary tumorigenesis in feral and inbred mice, our efforts have focused upon understanding the cellular basis of malignant progression in the mammary gland. Taking the point of view that mammary carcinomas arise as clonal populations of transformed tissue-specific stem cells and their differentiating progeny, we have initiated a long-term project aimed at elucidating the cellular, molecular, and genetic events underlying mammary epithelial cell growth, regeneration, and functional development. The reproductive capacity of the mammary epithelial stem cell is reduced coincident with the number of symmetric divisions it must perform. In a study of FVB/N mice with the transgene, WAP-TGFb1, we discovered that mammary epithelial stem cells were prematurely aged due to ectopic expression of TGF-b1. To test whether premature aging of mammary epithelial stem cells would have an impact on susceptibility or resistance to mammary cancer, female littermates from FVB/N X WAP-TGF-b1 mating were injected with mouse mammary tumor virus (MMTV) at 8-10 weeks of age. A total of 44 females were inoculated, maintained as breeders and observed for tumor development for up to 18 months. Only one mammary tumor appeared in 17 TGF-b1 females while 15 were collected from 29 wild type sisters. Premalignant mammary epithelial cells in infected glands were identified by transplantation of single cell (1x105) suspensions into nulliparous hosts and testing for hyperplastic outgrowth. Although the number of positive takes was significantly reduced with TGF-b1 cells, both MMTV-infected TGF-b1 and wild type cells produced hyperplastic outgrowths suggesting that premalignant transformation was achieved in each group. The results suggest a positive correlation between the procreative life-span of mammary epithelial stem cells and mammary cancer risk. In mice, rats and humans, a single early pregnancy provides lifelong reduction in mammary cancer risk. In rats and mice, the protective effect of pregnancy can be mimicked through hormonal application in the absence of gestation. This refractoriness to chemical induction of mammary tumorigenesis has recently been linked to the absence of a proliferative response in the parous epithelium when confronted with the carcinogen as compared with the nulliparous gland (Sivaraman et al., 2001; Sivaraman et al., 1998). Concomitant with the reduction in proliferative response is the appearance of stable activation of p53 in epithelial cell nuclei. This suggests that in response to the hormonal stimulation of pregnancy that a new cellular population is created with an altered response to carcinogen exposure. Employing the Cre recombinase/lox P system to identify mammary cells in situ, which have differentiated during pregnancy and expressed Cre from the whey acidic protein (WAP) promoter, a new parity-induced mammary epithelial cell population was discovered (Wagner, 2002). This population does not persist in nulliparous females at any age, but accumulates upon successive pregnancies in the mammary glands of parous females. The evidence shows that, in situ, these cells are committed to secretory cell fate and contribute extensively to the formation of secretory lobule development upon successive pregnancies. In this report, we demonstrate that these cells are pluripotent, i.e., capable of giving rise to all mammary epithelial subtypes and that they have an extended capacity to self renew over multiple transplant generations. We previously reported that TGFbeta less than =1 expressed from the WAP promoter reduces mammary cancer risk by promoting premature senescence in mammary stem cells (Boulanger & Smith, 2001; Kordon et al., 1995).